The proposed studies aim to develop high throughput protocols for assessing OP exposures by characterizing specific biomarker proteins in blood, and to make use of these more accurate measures of exposure to investigate gene/environment interactions related to genetic variability in the paraoxonase (PONI) gene, particularly with respect to OP exposures that occur during early development. Specific Aim 1 is to validate high throughput immunomagnetic bead (1MB) based isolation protocols for rapid purification and analysis of the active site peptides of two proteins, red cell acyl peptide hydrolase (APH) and plasma butyrylcholinesterase (BChE), as biomarkers of exposure to OP compounds. A targeted proteomics strategy based on microcapillary liquid chromatography-selected reaction monitoring-mass spectrometry (muLC-SRM-MS) will be validated and used to quantify the percentage modification of BChE and APH in archived samples from the Community Based Participatory Research Project. Data from the biomarker experiments will be correlated with other data available on urinary metabolite levels, inhibition of APH and/or BChE, and PONI status. Specific Aims 2 and 3 use knockout and humanized mouse models to determine the importance of PONI status for OP exposures that occur during critical periods of early development. Specific Aim 2 is to determine the extent to which APH and BChE are covalently modified following test exposure of pregnant mice to chlorpyrifos (CP) or CPO. Specific Aim 3 is to determine the extent to which BChE and APH are covalently modified following exposure of neonatal mice to CP or CPO. These aims will evaluate interactions among biomarkers of sensitivity, exposure and response in an in vivo system, allowing us to determine full dose responses of the OP compounds and generate quantitative biomarker data.